A family of enzymes have been described that exhibits cleavage specificity toward 5-hydroxymethylcytosine (5 hmC) over 5-methylcytosine (5 mC) and cytosine (C) (for example, WO 2011/091146, US 2012/0301881, Borgaro, et al., Nucleic Acids Research, 41(7):4198-4206 (2013), Wang, et al. Nucleic Acids Research, 39:9294-9305 (2011)). Representative members of this family of enzymes have been used for high resolution mapping of genomic 5 hmC in mouse embryonic cells (Sun, et al., Cell Reports, 3(2):567-576 (2013)). AbaSI described first in US 2012/0301881 has specificity for hmCN11-13/N9-10G, preferring 5-β glucosylhydroxymethylcytosine (5βghmC) over 5 mC in a ratio of 500:1 and 8000:1 with KOAc at a final concentration of 250 mM, but with a loss of 75% in the activity (Wang, et al. (2011)).
In mammalian genomic DNA, the most abundant modification is 5 mC. 5 hmC is only a small part relative to 5 mC, from 0-25% depending on the tissue. It is desirable for a reagent to have greater selectivity for 5βghmC converted from 5 hmC by a β glucosyltransferase with 100% efficiency over 5 mC to reduce the background digestion from 5 mC when determining modification. Although the family of enzymes that include AbaSI is a discriminator of 5βghmC over 5 mC and C, it would be desirable to enhance the discrimination between 5βghmC and 5 mC/C.